DESCRIPTION: Multiple ion channels influence neuronal excitability, and these are often subject to modulation by neurotransmitters. Prominent among these is a background or 'leak' K+ channel that is targeted for inhibition by neurotransmitters, leading to membrane depolarization and increased excitability. G protein-coupled receptors capable of mediating this effect have been identified for many transmitters (invariably those that couple via Gaq/l 1-family subunits), and whereas it represents a predominant mechanism for slow synaptic excitation throughout the brain, this phenomenon is particularly well described in motoneurons. Despite its widespread presence, the molecular identity of leak K+ channel(s) targeted for inhibition are unknown in most native systems, and the mechanisms of receptor-mediated channel inhibition remain obscure. A major goal of the current proposal is to identify the molecular substrate for a motoneuronal leak K about current. Evidence from our laboratory indicates that the two-pore domain K+ channel, TASK-1 (KCNK3), contributes to a pH- and neurotransmitter-sensitive leak K+ channel in hypoglossal motoneurons. New observations indicate that the closely related TASK-3 (KCNK9) subunit is also expressed in motoneurons. Moreover, preliminary data suggest that it may form heterodimers with TASK-1. We hypothesize that TASK-1 and TASK-3 form functional heterodimers that contribute to motoneuronal pH- and neurotransmitter-sensitive leak K+ currents. The second major goal is to characterize molecular mechanisms involved in receptor-mediated inhibition of these channels, focusing in turn on the molecules that represent the beginning (i.e., G proteins) and end points (TASK channels) of the receptor-activated signaling pathway. We hypothesize that Gag-family subunits provide the initial receptor-activated signal and that key determinants located in cytoplasmic domains of TASK channels are required for receptor-mediated TASK channel inhibition. For these studies, we utilize two experimental systems: a model system, based on heterologous expression of Gaq-coupled receptors and TASK channel subunits in mammalian cells, which recapitulates this modulatory mechanism; and a native neuronal system, in which heterologous gene expression is obtained in motoneurons using adenovirus vectors. The following Specific Aims are proposed: To determine if TASK channels can form functional heterodimers; To determine G protein subunits and channel domains involved in receptor-mediated TASK inhibition; and To determine contributions of TASK channels to motoneuronal currents and mechanisms of their modulation. These experiments will characterize molecular substrates underlying a native neurotransmitter-modulated leak K+ current and test key aspects of the mechanisms by which they are modulated.